Pro‐ and anti‐inflammatory cytokines mediate the progression of severe anemia in malaria‐infected children: A prospective study

Abstract Background Severe Plasmodium falciparum malarial anemia is still the principal cause of death in children in underdeveloped countries. An imbalance between proinflammatory and anti‐inflammatory cytokines is associated with malaria progression. This study evaluated circulating levels of selected inflammatory cytokines among malaria‐infected children in Ghana. Methods This case‐control study was conducted at Tamale Teaching Hospital, Ghana. One hundred and twenty children with malaria and 60 controls, aged 12−144 months were selected from April to July, 2023 for the study. Malaria was diagnosed through microscopy, full blood count was measured using hematology analyzer, and cytokines were measured using enzyme‐linked immunosorbent assay. Results Malaria‐infected children had higher tumor necrosis factor alpha (TNF‐α) (p < .001), interferon‐gamma (IFN‐ɣ) (p < .001), interleukin (IL)‐1β (p < .001), IL‐6 (p < .001), granulocyte macrophage‐colony stimulating factor (GM‐CSF) (p < .001), and IL‐10 (p < .001) levels than controls. Participants with high parasitemia had raised TNF‐α (p < .001), IFN‐ɣ (p < .001), IL‐1β (p < .001), IL‐6 (p < .001), GM‐CSF (p < .001), and IL‐10 (p < .001), but reduced IL‐3 (p < .001) and TGF‐β (p < .001) than those with low parasitemia. Severe malarial anemic children had elevated TNF‐α (p < .001), IFN‐ɣ (p < .001), IL‐1β (p < .001), IL‐6 (p < .001), GM‐CSF (p < .001), and IL‐10 (p < .001), but lower IL‐3 (p < .001) and TGF‐β (p < .001) than those with uncomplicated malaria. Conclusion Parasite density was the principal predictor of the cytokine levels, as parasitemia positively associated with IL‐10, GM‐CSF, IL‐6, IL‐1β, IFN‐ɣ, and TNF‐α, but negatively associated with IL‐3 and TGF‐β. Malaria is associated with enhanced secretion of pro‐ and anti‐inflammatory cytokines in Ghanaian children. Inflammatory cytokines may be involved in the development of severe malarial anemia in children. However, IL‐3 and TGF‐β may offer protection against severe malarial anemia.


| INTRODUCTION
Ghana is one of the developing countries heavily affected by malaria, and the situation worsens during the rainy season when mosquito bites are on the rise.The extensive interventions implemented over the years to control malaria in Ghana have had tremendous positive effects, with national prevalence in children declining by more than one-third from 26.7% in 2014 to 8.6% in 2022. 1 Despite this milestone in malaria control and prevention, malaria is still the principal cause of morbidity and mortality in Ghana, with children less than 5 years of age extremely affected. 2In Tamale, Northern Ghana, where malaria is holoendemic, Plasmodium falciparum is the principal cause of hematological and neurological disorders, and the leading contributor of hospitalization, especially in children. 3 Anemia is the most common complication of malaria in the tropics, and results from the related extensive sequestration of infected erythrocytes, lysis of erythrocytes, suppression of bone marrow, dyserythropoiesis, complement mediated, renal suppression of erythropoietin secretion, and excessive inflammatory response. 4,5The inoculation of plasmodium sporozoites into the subcutaneous layer of the human host and the associated complex life cycle of the parasite stimulate both innate and adaptive immunity responses. 6,7alaria-infected erythrocytes express P. falciparum erythrocyte membrane protein-1 (PfEMP-1), which enhances splenic and bone marrow sequestration of both parasitized and non-parasitized cells.Frequent sequestration of the cells in various organs disrupts the endothelium, triggers inflammation to release inflammatory cytokines.Proinflammatory cytokines contribute significantly to malaria protection and parasite elimination. 7,8During acute malaria, there is immediate secretion of pro-inflammatory T helper 1 (Th1) cytokines such as interleukin (IL)−1β, IL-6, interferon-gamma (IFN-ɣ) and tumor necrosis factor alpha (TNF-α), which retard parasite replication, enhance monocyte phagocytosis and promote elimination of infected red blood cells (RBCs). 6This tends to prevent the advancement from uncomplicated malaria to severe malaria and to prevent life-threatening complications.0][11][12] Th17 cells-mediated proinflammatory cytokines including IL-17 and IL-22 also play significant roles in immune response following plasmodium infection, by recruiting neutrophils, and triggering the release of other proinflammatory cytokines. 11Earlier studies in Ghana and Gabon reported increased IL-17 and IL-22 levels among children with plasmodium malaria. 11,12However, excess release of these proinflammatory cytokines during acute malaria may be detrimental to the host and cause severe complications such as severe anemia, organ damage and death. 6To modulate the secretion of proinflammatory cytokines and their associated complications in malaria, Th2 cell-mediated anti-inflammatory cytokines such as IL-10, IL-4, IL-13, and transforming growth factor-beta (TGF-β) are produced.Antiinflammatory cytokines control the humoral immunity, promoting parasite replication and clearance, and retard Th1 cytokine secretion. 13Disequilibrium between proinflammatory and anti-inflammatory cytokines results in noticeable adverse complications which are linked to malaria severity and an increased mortality rate especially in children. 13Hence, there is a need for a proinflammatory and anti-inflammatory balance to avert life-threatening complications associated with malaria.
Regardless of the clinical significance of pro-and anti-inflammatory cytokines in the host immune response against P. falciparum malaria, there is limited data among children in Northern Ghana.Therefore, this study assessed selected pro-and anti-inflammatory cytokines among malaria-infected children in Northern Ghana.The findings from this study will be beneficial in the management of severe malarial anemia in children.

| Ethical consideration and informed consent
This study was conducted in accordance with the Declaration of Helsinki on Research involving Humans.The Institutional Review Board of University for Development Studies, Ghana approved this study (UDS/IRB/15/ 2023).Permission was gotten from Tamale Teaching Hospital.Written informed consent was obtained from the parents or guardians of the participants by signing or thumb-printing the written informed consent form.

| Study participants
One hundred and twenty malaria-infected children, aged 12−144 months were recruited from April to July, 2023 at Tamale Teaching Hospital, Tamale, Ghana.Sixty apparently healthy age-and sex-matched children without malaria were selected from a Basic School in Tamale as controls.Tamale is the capital of the Northern Region, with a population of about 371,351 and is located at latitude 9.3930 N and longitude 0.8235 W. The hospital's digital address is NT-0101-5777.P. falciparum transmission is holoendemic and remains a major cause of morbidity and mortality in inhabitants of the area, with children being the most vulnerable. 3Major malaria transmission in Tamale occurs in the rainy season (April to June), and this area has benefited from the Seasonal Malaria Chemoprevention program implemented by the National Malaria Control Program, Ghana since 2015.This case-control study used Kelsey's formulae to determine sample size.Malaria parasitemia was graded into low parasite density (<1000 parasites/ µL), moderate parasite density (1000−10,000 parasites/ µL) and high parasite density (>10,000 parasites/µL). 11rticipants were further categorized based on the severity of anemia as mild/moderate malarial anemia (presence of malaria parasite and Hb ≥ 5.0 < 11.0 g/dL), and severe malarial anemia (presence of malaria parasite and Hb<5.0 g/dL). 14Age, sex and axillary temperature were collected from the participants' clinical records.Exclusion criteria included participants receiving anti-malarial drugs, comorbid with hemoglobinopathies, glucose-6-phosphate dehydrogenase deficiencies, human immunodeficiency virus, helminth infection, and any other disease that may promote inflammation or influence hematological parameters.Participants whose parents or guardians withheld their consent were excluded from the study.

| Laboratory assays
Thick and thin blood films were prepared from 6 and 2 µL, of dipotassium ethylenediaminetetraacetic acid (K 2 EDTA)-anticoagulated whole blood collected from each participant, respectively.The films were stained with 10% Giemsa working solution, examined under light microscope (Olympus) by two microscopy experts, and parasitemia was evaluated by multiplying the parasite count by the absolute leucocytes, and dividing by a set range of leukocytes (≥200 leukocytes). 14Full blood count (FBC) was measured using an automated hematology analyzer (URIT-5250, China).Malaria parasite identification and quantification, as well as FBC measurements, were performed immediately, and samples were collected daily at the Hematology Laboratory of the Tamale Teaching Hospital.To preserve bio-active TGF-β, platelet-poor plasma was collected from the EDTAanticoagulated blood on ice.The blood was centrifuged at 1000g for 15 min within 30 min of sample collection.Further centrifugation of the extracted plasma at 10,000g for 10 min at 4°C was done to ensure complete removal of platelet.Platelet count in the plasma was <10.0 × 10 9 /L for quality control purposes.The plateletpoor plasma was aliquoted, and stored at −20°C until analysis.Plasma levels of proinflammatory cytokines: TNF-α, IFN-ɣ, IL-1β, IL-6, granulocyte macrophagecolony stimulating factor (GM-CSF), and IL-3, and antiinflammatory cytokines: IL-10 and TGF-β were measured using sandwich enzyme-linked immunosorbent assay, with commercially prepared reagents (Biobase) at the University for Development Studies Laboratory, in accordance with manufacturer's instructions.The absorbance and corresponding concentrations of the cytokines were read at 450 nm using automated microplate reader (Poweam).

| Data analysis
IBM SPSS software version 26.0 was used to analyze the data.Normally distributed data were presented as mean ± standard deviation (SD), and skewed data were presented as median (1 st −3 rd quartiles).Categorical data were presented as frequencies with corresponding percentages, and compared using Pearson Chi-square.Continuous data between two categories were compared using Independent Student's T test (for parametric data) and Mann−Whitney U test (for non-parametric data).Plasma levels of inflammatory cytokines within three categories (parasitemia grading and anemia severity) were compared using the Kruskal−Wallis test.Multiple linear regression was used to examine age, sex, malaria status, parasite density and anemia severity for predictors of cytokine levels.Statistical significance was set at p < .05.

| Demographic, clinical, and hematological characteristics of the study participants
One hundred and twenty out of the 180 participants had the asexual form of P. falciparum in peripheral blood, and 60 were uninfected.The median age of the plasmodium-infected children and the controls were 24.0 (12.0−81.0)and 36.0 (12.0−84.0)months, respectively, but this was not statistically significant.About two-thirds of the participants were younger than 60 months with male percentage of 58.9%.The median parasite density was 440.0 (96.0−34,451.0)parasites/µL.

| DISCUSSION
The equilibrium between proinflammatory and antiinflammatory cytokines during P. falciparum malaria in children is crucial to prevent life-threatening complications in disease progression. 12This study evaluated selected pro-and anti-inflammatory cytokines among malaria-infected children in Northern Ghana.The significantly reduced hemoglobin concentration, RBC count and hematocrit among malaria-infected children compared to uninfected participants observed in this study is consistent with earlier findings reported in Ghana [15][16][17] and other developing countries. 10,18,19The extensive hemolysis of parasitized and non-parasitized red cells, extreme sequestration of erythrocytes in organs, direct interaction with plasmodium surface antigens, associated dyserythropoiesis, related complement activation, bone marrow suppression, and the contribution of inflammatory mediators could account for the occurrence of anemia in the malaria-infected participants. 4,5,9,202][23] The reduced platelet count associated with malaria may be due to enhanced sequestration by the spleen, immune-mediated destruction of thrombocytes and associated ineffective hemopoiesis. 21,23,24The reduced lymphocyte count among malaria infected participants observed in this study has also been reported in Ghana, and related it to the associated Fas-induced apoptosis leading to acute destruction of lymphocytes. 21Jiero et al. reported monocytosis in acute malaria as monocytes are involved in phagocytic activities to enhance parasite clearance. 25However, the current study observed comparable monocyte counts  between malaria infected and uninfected participants.The variation in the outcomes may be due to factors including disease severity, parasitemia, comorbidity and host immunity. 25ven though there have been efforts to modify the inflammatory cytokine profile associated with severe malaria in the past via adjunct therapies, this has not yielded the expected clinical outcome.This study found increased plasma levels of proinflammatory cytokines: TNF-α, IFN-ɣ, IL-1β, IL-6, and GM-CSF, and the antiinflammatory cytokine IL-10 in malaria-infected children than the controls without malaria, and the levels were higher in participants with high parasitemia.7][28][29] During the erythrocytic phase of malaria pathogenesis, P. falciparum-erythrocytes express PfEMP-1 surface antigen which facilitates the adherence of infected erythrocytes to endothelial surfaces by binding to endothelial cell adhesion molecules such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, P-selectin and E-selectin. 8The PfEMP-1-induced extensive sequestration of parasitized and nonparasitized erythrocytes to endothelial surfaces enhance excessive inflammatory response with subsequent release of proinflammatory cytokines, such as IFN-ɣ, TNF-α, IL-6 and IL-1β. 8,9The enhanced production of proinflammatory cytokines mainly controls the growth of the plasmodium and enhances parasite elimination to inhibit the progression to severe malaria, but unregulated elevation of the cytokines may be detrimental to the host. 6imilarly, children with malaria had relatively raised plasma levels of IL-10, which is comparable with other studies. 4,10,26,28,30Interleukin 10 is an anti-inflammatory and a pleiotropic cytokine that inhibits inflammation by directly reducing the release of proinflammatory cytokines such as TNF-α and IFN-ɣ. 31,32However, levels of TNF-α, 10 IL-1β, 16 IL-6, 8,33 IFN-ɣ, 33 IL-10, 11,33 and GM-CSF 27 were not different when malaria-infected children were compared to their counterparts without the infection in previous studies.The difference in the findings remain unclear, but could be linked to factors such as degree of parasitemia, immunity of the host and disease severity. 8,10,11,27lthough plasma levels of IL-3 and TGF-β were similar between children with malaria and the uninfected group, the levels of the cytokines were lower in participants with hyperparasitemia in the present study, and this observation is similar to earlier findings. 32,34,35In a study by Meyer et al. that recruited 1015 Ghanaian P. falciparum-infected children at the age of 3 months and followed them up to 2 years, transient increase in IL-3 was observed among the children with acute P. falciparum malaria, but the levels declined as the disease progressed.The IL-3 single nucleotide  polymorphisms rs40401 TT and rs40401 CT were observed to provide protection against malaria attacks. 35Transforming growth factor-β is a potent anti-inflammatory and pleiotropic cytokine that inhibits the inflammatory response and regulates malaria pathogenesis, serving as a protective mechanism against severe infection. 36n the current study, participants with malaria had higher TNF-α/IL-10, TNF-α/TGF-β, IFN-ɣ/TGF-β, and IL-1β/TGF-β, but lower IFN-ɣ/IL-10, IL-1β/IL-10, and IL-6/TGF-β ratios than those in the control group.On the contrary, TNF-α/IL-10 and IL-6/IL-10 ratios were reduced when malaria-infected children were compared with uninfected group in Malawi. 37Again, a study by Frimpong et al. among Ghanaian children reported that patients with malaria had comparable ratios of IFN-γ/IL-10, TNF-α/IL-10, and IL-6/IL-10 than controls without malaria. 11Geographical variations may account for the variations in the findings.Whiles the current study was conducted in Northern Ghana, the Frimpong et al. study recruited participants residing in Accra, a coastal area in the country.
In the present study, children with severe malarial anemia had significantly elevated plasma levels of TNF-α, IFN-ɣ, IL-1β, IL-6, GM-CSF and IL-10 than those with uncomplicated malaria, and this is in consonance with earlier findings. 4,26,27Even though inflammatory cytokines are essential for inhibiting plasmodium parasite growth and elimination, excessive production negatively affects physiological processes and causes lifethreatening complications such as severe anemia. 6NF-α has been recognized as a potent inhibitor of erythropoiesis, by retarding erythroid progenitor cell proliferation in humans through the inhibition of erythropoietin synthesis. 38In addition, TNF-α blocks iron recycling from macrophages, restricting iron to storage sites and initiating the development of functional iron deficiency anemia.Again, TNF-α, independent of hepcidin, blocks the absorption of iron from the duodenum and jejunum by downregulating the expression of divalent metal transporter-I, increasing the deposition of ferritin in the gastrointestinal tract and reducing iron available for erythropoiesis. 39The role of IL-1β in the development and pathological etiology of anemia has been reported. 40Interleukin-1β negatively influences iron metabolism preventing iron's utilization for hemoglobin synthesis.In addition, IL-1β suppresses the renal release of erythropoietin, interferes with erythropoietin receptors and eventually promotes early apoptosis of erythroid progenitors. 40Despite the effective role of IFN-ɣ in promoting plasmodium parasite clearance, the proinflammatory cytokine may retard erythropoietic activities by suppressing the proliferation and differentiation of red cell progenitors. 41This eventually leads to early apoptosis of erythroblasts with subsequent reduction in red cell parameters in peripheral blood. 93][44] IL-6 induces the release of hepcidin which inhibits the unique cellular iron transporter ferroportin, prevents intestinal iron absorption, interferes with the release of iron from senescent red cells and restricts iron (ferritin) to storage sites.This phenomenon suppresses erythropoiesis through an insufficient supply of iron to the bone marrow and subsequently leads to functional iron deficiency anemia. 43,44Independent of iron restriction, IL-6 has been reported to directly repress erythropoietindependent TF-1 erythroid maturation. 45The contribution of GM-SCF in the pathogenesis of severe malarial anemia has been emphasized as the proinflammatory cytokine has been reported to inhibit erythropoiesis by interfering with erythroblastic island formation via macrophages, suppressing the proliferation and differentiation of early erythroid progenitors. 46On the other hand, children with severe malarial anemia had lower IL-3 and TGF-β levels than those with uncomplicated malaria in the current study, and this is similar to previous findings. 33,35The protective role of IL-3 against severe malarial anemia has been reported, as the cytokine principally functions to modulate the production of various blood cells by stimulating the proliferation and differentiation of both early pluripotent stem cells and committed hemoblasts. 47IL-3, in unison with other signaling molecules, such as erythropoietin, IL-6 and GM-CSF induces the differentiation of multipotent hemopoietic stem cells into the myeloid progenitor lineage, but in synergy with IL-7 promotes lymphoid progenitor generation. 48TGF-β is a special protein that critically plays a role in hemopoietic stem cell regulation, with downregulation of its expression in diseases including malaria related to the development of severe anemia. 49,50he relative contributions of age, sex, malaria status, parasite density and anemia severity as predictors of cytokine levels were assessed using multivariate linear regression analysis.Parasite density was observed to be the strongest predictor of all cytokine levels.Parasite density positively associated with IL-10, GM-CSF, IL-6, IL-1β, IFN-ɣ, and TNF-α, but negatively associated with IL-3 and TGF-β.8][29] High parasite density is associated with enhanced cytoadherence of both P. falciparum-infected and uninfected erythrocytes to the endothelium.7][28][29] The negative association between parasite density, and IL-3 and TGF-β is due to the ability of plasmodium parasites to suppress the release of these two cytokines from immune cells such as monocytes, CD4+ and CD8+ cells. 34

| Major strength and limitations
This study revealed the relationship between severe malarial anemia and inflammatory cytokines in P. falciparum-infected children.Findings from the current study were restricted to children aged 1-12 years.In addition, the study could not assess other important proand anti-inflammatory cytokines.Also, the study could not perform molecular assessment to identify submicroscopic malaria infection.

| CONCLUSION
Malaria presents with enhanced release of TNF-α, IFN-ɣ, IL-1β, IL-6, GM-CSF, and IL-10, but downregulates IL-3 and TGF-β in Ghanaian children.Inflammatory cytokines may contribute to the development of severe malarial anemia in children.However, IL-3 and TGF-β may offer protection against severe malarial anemia and prevent further complications.Therapeutic protocols should target pro-and anti-inflammatory cytokines for the management of severe malarial anemia in children.Future genotypic studies on cytokines in malaria are recommended.

T A B L E 5
Relationship between inflammatory cytokines and degree of parasitemia among malaria-infected participants.

T A B L E 6
Relationship between inflammatory cytokines and anemia severity among malaria-infected participants.
The data are presented as median (interquartile range), and compared using the Kruskal−Wallis test.p < .05 was considered statistically significant.Multivariate regression analysis to determine predictors of cytokine levels.